The herpes simplex virus (HSV) DNA polymerase (pol) has been shown to be of central importance for viral DNA replication (3,4,5,18) and is one of the major target enzymes of current antiviral chemotherapy. The nucleotide sequences of the HSV-1 and HSV-2 DNA polymerases have been determined in several laboratories (8,12,15,20,23). The sequences suggest that the HSV-1 gene encodes a polypeptide of 1235 amino acids, approximately 136 kDa (8,12,20), and polypeptides of this size have been associated with HSV-specific DNA polymerase activity in infected cells (18,19,22), by in vitro translation (6), and in a yeast expression system (10). The HSV-2 enzyme is 1240 amino acids in length with a molecular weight of 137 kDa and shares 95.5% amino acid sequence homology to the HSV-1 enzyme.
Comparison of the deduced amino acid sequence of the HSV DNA polymerase with other herpesvirus and eukaryotic DNA polymerases (2,8,9, 14,15,26,27) reveals several protein domains which are conserved among the different pol enzymes. The most conserved regions are clustered towards the carboxy terminus. These conserved regions are generally thought to be involved in substrate recognition or catalysis by the DNA pol enzymes (8,9,26,27). In spite of the apparent structural similarities between the mammalian and viral enzymes, the HSV polymerases can readily be distinguished by their enhanced activity in the presence of high salt concentrations and greater sensitivity to such agents as acycloguanosine triphosphate (ACVTP, acyclovir triphosphate) and phosphonoacetate (PAA) (7,17). This suggests that there are some structural differences in the binding sites between these molecules and selective drug development relies, at least in part, upon these subtle differences.
Consistent with comparative sequence analysis, precise mapping of drug resistance and temperature sensitive mutations (4,5,13,14,24) have further suggested that the active site of the enzyme maps within the carboxy terminus of the pol protein, specifically between amino acid residues 597-1008.
In agreement with the functional assignment, Weisshart and Knopf (25) have shown that antibodies raised to the central and the C-terminal portions of the HSV-1 protein inhibited the polymerizing activity by 70-90%, respectively. In contrast, antibodies directed towards the N-terminal HSV-1 domain did not neutralize the enzymatic activity. In addition, amino terminal deletions have been shown to retain HSV-1 DNA pol activity, while carboxy terminal deletions can inactiviate the enzyme (6). Thomas et.al. (22) demonstrated that an antisera to a non-conserved N-terminal peptide (residues 1-15) could immunoprecipitate HSV-1 DNA polymerase activity, but they did not discuss the effect of that antisera on neutralization of enzymatic activity.
The present invention demonstrates that antisera to two distinct carboxy terminal peptides (HSV-1 residues 1100-1108 and 1216-1224) can specifically neutralize HSV-1 and HSV-2 polymerase activity. These peptide sequences are unique to HSV and are not conserved in other herpes viruses or eukaryotic polymerases.